The mechanisms of enzyme secretion is studied using as models the formation and secretion of invertase by the eukaryote yeast (Saccharomyces) and of penicillinase by the prokaryote Bacillus licheniformis. The subunit structure of the mannan-protein form of investase and of the internal carbohydrate-free enzyme will be compared to clarify their biosynthetic relationship and invertase located in actively-secreting protoplasts by immunological electron microscopy. The alkaline phosphatase present in the yeast vacuole will also be isolated and characterized since it appears to be a glycoprotein but its synthesis, in contrast to other glycoproteins, is not sensitive to the antibiotic tunicamycin. Studies on penicillinase will emphasize a search for additional membrane phospholipoproteins with phosphatidylserine-containing hydrophobic tails in Bacilli (other Bacillus penicillinases will also be isolated and characterized), and the releasing enzyme present in mesosomes and apparently responsible for the physiological cleavage of membrane penicillinase to yield exoenzyme will be purified and characterized. Negative mutants will be sought to confirm its apparent requirement for the secretion process. The phosphatidylseryl tRNA found in the membranes of B. licheniformis will be examined to determine whether more than a single isoaccepting seryl tRNA can be converted to the phosphatidylseryl form. The in vitro penicillinase synthesizing system will be further developed to obtain chain initiation with the goal of determining at what point the phosphatidylseryl residue becomes a part of the peptide chain. BIBLIOGRAPHIC REFERENCES: Bettinger, G.E., and Lampen, J.O. Further evidence for a partially-folded intermediate in penicillinase secretion by Baccilus licheniformis. J. Bacteriol. 121: 83-90 (1975). Colonna, W.J., Cano, F.R., and Lampen, J.O. Microheterogeneity in yeast invertase. BBA 386: 293 - 300 (1975).